The long-range goal of this research is to understand how endochondral calcification, a process vital for skeletal development and fracture healing, works. Because isolated matrix vesicles (MV) initiate calcification by acquiring large amounts of Ca2+ and Pi, they make an excellent model on which to base the fabrication of clinically useful biomaterials for stimulating bone healing. Major progress was made during the past grant period in characterizing the mechanism of MV mineralization. We have identified and characterized the Ca2+ transporter, made progress in purifying the MV phospholipase A2 and have laid important groundwork in cloning the Pi transporter. We have uncovered important information on MV lipid metabolism and in characterizing the initial mineral phase of MV, and have done substantial pilot work on reconstitution of the nucleational core and formation of synthetic MV. Building on these findings, our major goals for the next grant period are to elucidate how these lipids, proteins and electrolytes interact to trigger MV formation and mineralization. Since we have already characterized the Ca2+ porter, lipid, and electrolyte components of the nucleational complex, we will now characterize the MV Pi ion porter. Based on the finding of major changes in lipid composition during MV mineralization, we will now characterize two enzymes that appear to be responsible for these changes. During this past grant period we have also acquired a large body of spectroscopic data on the nature of the nucleational complex. We will now use this data to guide us in our reconstitution of the synthetic complex, as well as to deduce its structure using molecular simulations. Collectively, this body of information will allow us to precisely construct an implantable biomimetic material for stimulating bone healing. Our specific aims are: 1) to characterize three new MV proteins that appear to be critical to MV function, 2) to further elucidate MV formation, minerals, and metabolism, and 3) to reconstitute and simulate MV structure and function. Specifically we will characterize critical interactions that occur between the annexins, PS, Ca2+ and Pi during MV formation. Also the nature of the first mineral phase formed by MV and changes in MV lipid composition that accompany mineralization will be examined. The MV Na+-dependent Pi-transporter, the phospholipase A2 and the phospholipid base-exchange enzyme will be identified, cloned and sequenced, and their properties characterized. The MV nucleational complex will then be synthesized, characterized, and modeled by computer simulation. Finally, with this information in hand, functional MV will be reconstituted and tested for bone-healing properties.